We propose to investigate the role of actin in the control of red cell shape and membrane deformability of both normal and abnormal red cells by using biochemical and biophysical techniques to study the systematic recombination of oriented red cell membranes and purified membrane proteins. We will characterize the association of monomeric and filamentous actin with the human erythrocyte membrane by using 3H-actin and a quantitative polymerization assay involving (32P-ATP)-3H-actin which will enable us to detect and quantify membrane nucleation sites for actin polymerization. Peripheral membrane proteins, or protein complexes, which we have shown in preliminary studies to stimulate the binding of G actin to inside out vesicles will be identified by fractionation of soluble proteins by a number of methods. Integral membrane proteins which we have implicated in the binding of F-actin to red cell membranes will be identified by solubilization of membranes in non-ionic detergents and rate zonal sedimentation in the presence of F, or G actin. In addition, we will investigate the assembly of cytoskeletal analogues consisting of spectrin, actin and minor membrane proteins such as band 4.1. The morphology of these protein complexes will be examined by electron microscopy of low angle shadowed and negative stained preparations. Biochemical characterization will rely upon a novel quantitative micro-assay of actin polymerization in addition to standard methods such as column chromatography and rate zonal centrifugation. The significance of spectrin phosphorylation and aggregation state (i.e. dimeric or tetrameric spectrin) to such associations will be investigated. We will investigate the association of actin with spectrin and membranes of abnormal red cells including hereditary spherocytes, eliptocytes and poikilocytes.